Compositions and methods for assessing gut function

ABSTRACT

Compositions and methods for assessing gut function are disclosed. They are designed to provide accurate, rapid, point-of-care or in-community assessment of enteric dysfunction.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/200,311 filed on Nov. 26, 2018, which is a divisional applicationof U.S. application Ser. No. 14/538,770 filed on Nov. 11, 2014, whichclaims the benefit of U.S. Provisional Application No. 61/902,637,entitled “Compositions and Methods for Assessing Gut Function,” filed onNov. 11, 2013, the entire contents of each are hereby incorporated byreference.

BACKGROUND Field

The present disclosure relates to compositions and methods for assessinggut function, more specifically to noninvasive methods for assessing gutbarrier function.

Related Art

Gut mucosal integrity is a major challenge facing clinical medicine.Proper functioning of the intestines depend on the mucosa havingsufficient surface capacity with which to absorb nutrients as well hashaving sufficient structural integrity to maintain the barrier functionof the lining of this organ.

Abnormally increased gut permeability quite likely plays a role ininflammatory bowel diseases (IBD) (i.e., Crohn's disease and ulcerativecolitis) and identifying abnormally increased gut permeability can bevery helpful in diagnosing disease, as well as monitoring and adjustingtherapy directed against gut inflammation. Identifying increased gutpermeability might assist in the management of a variety of disordersthat are not directly manifest as intestinal dysfunction. Thesedisorders non-alcoholic fatty liver disease, cirrhosis of the liver,arthritis, diabetes, and irritable bowel syndrome.

Various molecular strategies to probe the structure and function of thesmall bowel have been developed. The chief goal of all of thesestrategies is to directly or indirectly assess the structural integrityof the intestinal epithelium, which is comprised of a carpet of highlyspecialized columnar epithelial cells joined by tight junctions andadherens junctions. Molecules are selected to probe the competency ofuptake and of permeability. Generally, integrity lesions that result inincreased passive diffusion across the mucosa are detected bychallenging a host with a substance that is not found in the diet, andassessing its uptake and/or clearance by studying the blood and theurine. In this model, uptake and excretion are abnormal. Conversely, guttransport capacity is assessed by administering a challenge substancethat is easily absorbed in health and disease, but where uptake islimited by surface availability (as would be hindered in anatomic shortgut or diminished villous surface area). Such substances are thenmeasured in the urine and blood, and uptake reflects a gut ofappropriate mass.

The most widely used test is termed the lactulose to mannitol ratio(L:M). Sugars (i.e., lactulose and mannitol) are administered orally,and their excretion is measured in the urine. The basis for the testlies in their two differential molecular weights. The larger sugar,lactulose, MW=342, is minimally absorbed during transit through anintact gut lined by intact epithelial, with highly competent tightjunction functionality, and this disaccharide is therefore considerednearly impermeant. The smaller sugar, mannitol (MW=182), in contrast, isassimilated by an intact as well as an injured (permeant) gut viatranscellular pathways and this absorption is proportional to theabsorptive capacity of the gut. Both sugars distribute throughout thebody in a hydrophilic compartments, and are then cleared by glomerularfiltration. They are then measured in tandem in the urine. Theco-administration, and the use of ratios in the urine, obviate singlemolecule assessments, because the ratio is independent of gastricemptying, or partial vomiting of the challenge sugars. Other sugars havebeen used to measure small and large bowel permeability, and includerhamnose and sucralose and D-xylose.

Measurements of challenge substances can be technically difficult. Ifpeak or repeated circulating values are sought, phlebotomy is required,and there are assumptions about the kinetics of the peak. A urinecollection is less invasive, but usually occurs over multiple hours, andis cumbersome to perform. An indirect test, such as measuring bacterialmetabolism reflecting undigested sugar, requires specialized equipment.Hence, specimen acquisition, handling, and analysis are all verycritical, but difficult to perform.

Thus, there remains a need for improved methods and compositions forassessing gut function.

SUMMARY

Accordingly, the inventors herein disclose new compositions and methodsfor assessing gut function. The methods are designed to be noninvasive,sensitive, and convenient.

Provided is a composition for assessing gut function, and mostspecifically barrier integrity, in a subject in need thereof, comprisinga fluorescent challenge molecule, wherein the fluorescent challengemolecule is not substantially absorbed by a healthy gut.

Provided is a composition for assessing gut function in a subject inneed thereof, comprising at least two fluorescent challenge molecules,wherein one fluorescent challenge molecule is not substantially absorbedby a healthy gut, and the other is substantially absorbed by a healthygut. The second molecule may possess different photophysical propertiesfrom the first challenge molecule. Thus, the two molecules displaydifferent absorption and emission wavelengths, and allow real-timemeasurement of uptake and permeability in the gut.

Provided is a method for assessing gut function in a subject in needthereof, comprising the steps of: administering an effective amount of acomposition of a fluorescent challenge molecule, wherein the fluorescentchallenge molecule is not substantially absorbed by a healthy gut intothe subject's gut; irradiating the composition absorbed by the subject'sgut with non-ionizing radiation, wherein the radiation causes thecomposition to fluoresce; detecting the fluorescence of the composition;and assessing gut function in the subject based on the detectedfluorescence.

Provided is a method for assessing gut function in a subject in needthereof, comprising the steps of: administering an effective amount of acomposition comprising two fluorescent challenge molecules—a fluorescentchallenge molecule that is not substantially absorbed by a healthy gut,and a second fluorescent challenge molecule is substantially absorbed bya healthy gut—into the subject's gut; irradiating the compositionabsorbed by the subject's gut with non-ionizing radiation, wherein theradiation causes the composition to fluoresce; detecting thefluorescence of each fluorescent challenge molecule in the composition;and assessing gut function in the subject based on the detectedfluorescence. GUT function is assessed by comparing the detectedfluorescence of the fluorescent challenge molecule that is notsubstantially absorbed by a healthy gut to the fluorescent challengemolecule that is substantially absorbed by a healthy gut. The twofluorescent challenge molecules may possess different photophysicalproperties allowing for the simultaneous measurement of the fluorescencethe two molecules.

Provided is a kit for assessing gut function in a subject in needthereof, comprising: a composition comprising one or more orallyadministered fluorescent challenge molecules, wherein the fluorescentchallenge molecule(s) is not substantially absorbed by a healthy gut,and written instructions for assessing gut function in the subject,comprising the steps of: administering an effective amount of thecomposition into the subject's gut; irradiating the composition absorbedby the subject's gut with non-ionizing radiation, wherein the radiationcauses the composition to fluoresce; detecting the fluorescence of thecomposition; and assessing gut function in the subject based on thedetected fluorescence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a fluorescent challenge molecule, whereinthe fluorescent dye is conjugated to mannitol;

FIG. 2 shows anticipated fluorescence measurement data for permeable andnon-permeable fluorescent challenge molecules administered to a subject;

FIG. 3 shows Plasma Concentrations vs. Time Curves of Example 1 inSprague-Dawley Rats Following Single Dose of Example Compound 1 givenIntravenously and Orally;

FIG. 4 shows the emission spectra of Example Compound 2;

FIG. 5 shows Example Compound 3 clears from the body via the renalsystem;

FIG. 6 shows anticipated Fluorescence vs. Time Curves for compositionscomprising both challenge molecules that are substantially and notsubstantially absorbed in healthy guts and diseased guts;

FIG. 7 shows an anticipated Fluorescence vs. Time Curve for compositionscomprising both challenge molecules that are substantially and notsubstantially absorbed in a gut having a proximal injury; and

FIG. 8 shows an anticipated Fluorescence vs. Time Curve for compositionscomprising both challenge molecules that are substantially and notsubstantially absorbed in a gut having a distal injury.

DETAILED DESCRIPTION Abbreviations and Definitions

To facilitate understanding of the disclosure, a number of terms andabbreviations as used herein are defined below as follows:

When introducing elements of the present disclosure or the preferredembodiment(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “and/or” when used in a list of two or more items, means thatany one of the listed items can be employed by itself or in combinationwith any one or more of the listed items. For example, the expression “Aand/or B” is intended to mean either or both of A and B, i.e. A alone, Balone or A and B in combination. The expression “A, B and/or C” isintended to mean A alone, B alone, C alone, A and B in combination, Aand C in combination, B and C in combination or A, B, and C incombination.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of a compound, dose, time, temperature, and the like,is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1%of the specified amount.

The term “gut” as used herein designates the gastrointestinal ordigestive tract (also referred to as the alimentary canal) and it refersto the system of organs within multi-cellular animals which takes infood, digests it to extract energy and nutrients, and expels theremaining waste.

The term “gut function” as used herein refers to any aspect of thefunctioning of the gut and/or structural integrity of the gut. Forexample, the methods of the present disclosure may be used to provide anindication or assessment of: the absorptive capacity of the gut; theability of the gut to absorb nutrients; the permeability of the gut; theability of the gut to hydrolyze compounds; the surface area of the gutand/or small intestine; the functional surface area of the gut and/orsmall intestine; the barrier function of the gut; damage to the gut; thedegree of mucosal damage in the gut; damage to the villi in, rater alia,the small intestine; the villous height of the brush border of the smallintestine; the presence of any disease or pathology which is associatedwith a change in gut function as described herein; the presence of aninflammatory condition; the presence of a infection; the response totreatments to correct any or all of the above lesions, as well as anyother aspect of gut functioning or gut structural integrity that wouldbe evident to one of skill in the art.

The term “fluorescent dye” as used herein refers to a dye that absorbslight at a first wavelength and emits at second wavelength that islonger than the first wavelength.

The term “fluorescence” refers to luminescence in which the molecularabsorption of a photon triggers the emission of another photon with alonger wavelength.

The term “challenge molecule” refers to a compound to be administered toa subject to observe the physiological response that occurs. The“challenge” may be, for example, introducing a compound to a subject toassess the gut. This may be one that is not normally absorbed by ahealthy gut, wherein absorption may represent permeability of the gut.

The terms “degradation” and “digestion” as used herein refer to thechemical breakdown of a compound to release e.g. its component atoms ora simpler compound.

The term “carbohydrate” as used herein is defined to include polyhydroxyaldehydes, or polyhydroxy ketones or substances that yield suchcompounds on hydrolysis. The term “carbohydrate” includesmonosaccharides, oligosaccharides, disaccharides, trisaccharides,tetrasaccharides, pentasaccharides, hexasaccharides, polysaccharides,homopolysaccharides, and heteropolysaccharides. The term includes any ofthe aldoses, as well as glucose, dextrose, mannose, galactose arabinose,xylose, ribose, fructose, sucrose, altrose, allose, idose, gulose,talose, lyxose, threose, erythrose, apiose, and any of the same in acidform. The term also includes deoxy sugars and deoxy-aldoses, includingrhamnose and fucose. The term further includes glyceraldehyde,cellulose, starch, glycogen, and amylose. The term also includescarbohydrate derivatives, such as acetals, ketals, acyl esters and thelike.

The term “non-ionizing radiation” refers to lower-energy radiation, suchas visible light, near infrared, infrared, microwaves, and radio waves.The ability of an electromagnetic wave (photons) to ionize an atom ormolecule depends on its frequency. Radiation on the short-wavelength endof the electromagnetic spectrum—x-rays, and gamma rays—is ionizing.Therefore, when using the term “non-ionizing radiation” it is intendedto mean electromagnetic waves having a frequency not sufficient toionize an atom or molecule.

The phrase “not substantially absorbed” is intended to refer tomolecules that may be distinguished by the difference in their gutabsorption in a healthy versus a diseased or injured gut. In certainembodiments, not substantially absorbed means less than 99% of theadministered amount is absorbed. In certain embodiments, notsubstantially absorbed means less than 95% of the administered amount isabsorbed. In certain embodiments, not substantially absorbed means lessthan 90% of the administered amount is absorbed. In certain embodiments,not substantially absorbed means less than 80% of the administeredamount is absorbed. In certain embodiments, not substantially absorbedmeans less than 70% of the administered amount is absorbed. In certainembodiments, not substantially absorbed means less than 60% of theadministered amount is absorbed. In certain embodiments, notsubstantially absorbed means less than 50% of the administered amount isabsorbed. In certain embodiments, not substantially absorbed means lessthan 40% of the administered amount is absorbed. In certain embodiments,not substantially absorbed means less than 30% of the administeredamount is absorbed. In certain embodiments, not substantially absorbedmeans less than 20% of the administered amount is absorbed. In certainembodiments, not substantially absorbed means less than 10% of theadministered amount is absorbed. In certain embodiments, notsubstantially absorbed means less than 5% of the administered amount isabsorbed. In certain embodiments, not substantially absorbed means lessthan 1% of the administered amount is absorbed. The phrase “notsubstantially absorbed,” when describing compositions, methods and kitscomprising more than one fluorescent challenge molecule, refers tocombinations of challenge molecules that may be distinguished by thedifference in their gut absorption in a healthy versus a diseased orinjured gut. In certain embodiments, the ratio of each challengemolecule's gut absorbance is greater than about 1. In certainembodiments, the ratio of each challenge molecule's gut absorbance isgreater than about 2. In certain embodiments, the ratio of eachchallenge molecule's gut absorbance is greater than about 3. In certainembodiments, the ratio of each challenge molecule's gut absorbance isgreater than about 4. In certain embodiments, the ratio of eachchallenge molecule's gut absorbance is greater than about 5. In certainembodiments, the ratio of each challenge molecule's gut absorbance isgreater than about 10. In certain embodiments, the ratio of eachchallenge molecule's gut absorbance is greater than about 20. In certainembodiments, the ratio of each challenge molecule's gut absorbance isgreater than about 30. In certain embodiments, the ratio of eachchallenge molecule's gut absorbance is greater than about 40. In certainembodiments, the ratio of each challenge molecule's gut absorbance isgreater than about 50.

The term “substantially simultaneously,” as used herein, is intended tomean simultaneously or approximately simultaneously and is meant toencompass variations of 15%, 10%, 5%, 1%, 0.5%, or even 0.1% of thespecified time of occurrence.

The term “substantially later in time” as used herein, is intended tomean not simultaneously, and, is meant to encompass time differences ofgreater than 15%, 20%, 30%, or even 50% of the specified timedifference, or a difference that is statistically significantly later intime.

Compositions

The present disclosure provides a composition for assessing gut functionin a subject in need thereof, comprising a fluorescent challengemolecule, wherein the fluorescent challenge molecule is notsubstantially absorbed by a healthy gut. Additionally, a secondfluorescent challenge molecule, a control molecule that may be absorbedin health and disease, and thereby serve as a normalizing determinationagainst which the larger molecule's uptake will be measured. Thesecompositions may be measured within a subject utilizing noninvasivetechniques, and provide real time data and results.

The fluorescent challenge molecule may be a fluorescent dye. Thefluorescent dyes of the present disclosure tend to have absorption,excitation, and emission wavelengths that are all within thenear-infrared (NIR) or visible spectrum of about 350 nm or greater. Thisis beneficial for diagnostic procedures since visible and NIR light arenot likely to damage tissue. In contrast, ultraviolet (UV) light thathas a wavelength of less than about 350 nm can damage tissue. Lighthaving a wavelength of about 350 nm or greater tends to penetrate intotissues thereby permitting diagnostic procedures to be conducted intissues of interest that may not be reachable using UV wavelengths thatare less than about 350 nm. Suitable fluorescent dyes include acridines,acridones, anthracenes, anthracylines, anthraquinones, azaazulenes, azoazulenes, benzenes, benzimidazoles, benzofurans, benzoindocarbocyanines,benzoindoles, benzothiophenes, carbazoles, coumarins, cyanines,dibenzofurans, dibenzothiophenes, dipyrrolo dyes, flavones,fluoresceins, imidazoles, indocarbocyanines, indocyanines, indoles,isoindoles, isoquinolines, naphthacenediones, naphthalenes,naphthoquinones, phenanthrenes, phenanthridines, phenanthridines,phenoselenazines, phenothiazines, phenoxazines, phenylxanthenes,polyfluorobenzenes, purines, pyrazines, pyrazoles, pyridines,pyrimidones, pyrroles, quinolines, quinolones, rhodamines, squaraines,tetracenes, thiophenes, triphenyl methane dyes, xanthenes, xanthones,and derivatives thereof. In certain embodiments, the fluorescent dye isa pyrazine.

The uptake of larger molecules reflects gut porosity. Thus, thefluorescent challenge molecule is relatively large. Specific embodimentsinclude3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyrazineand 3,6-N,N′-bis(2,3-dihydroxypropyl)-2,5-pyrazinedicarboxamide.

The fluorescent challenge molecule may be a fluorescent dye conjugatedto a carbohydrate. Suitable carbohydrates include large molecular weightcarbohydrates such as lactulose (MW=342), sucrose (MW=342), mannitol(MW=180), and sucralose (MW=398). There is some site specificity:lactose and lactulose reflect porosity in the small bowel, sucrosereflects porosity in the stomach, and sucralose reflects porosity in thelarge bowel. In certain embodiments, the carbohydrate is lactulose.

The fluorescent challenge molecule may be a fluorescent dye conjugatedto polyethylene glycol (PEG). Polyethylene glycol refers to oligomersand polymers with a molecular mass below 20,000 g/mol. As referred toherein, a “PEG unit” means a —CH₂CH₂O— unit. PEG units are typicallycomponents of highly soluble oligomers and polymers of ethylene glycol.Further, they tend to be highly biocompatible, non-immunogenic, andnon-toxic.

The composition may include a second fluorescent challenge molecule,wherein the fluorescent challenge molecule is substantially absorbed bya healthy gut. The second challenge molecule may be similar to the firstchallenge molecule, in that it may include a fluorescent dye; however,the second molecule is designed to be absorbed by the gut. In certainembodiments, the second fluorescent challenge molecule is3,6-diaminopyrazine-2,5-dicarboxylic acid.

The second challenge molecule may also include a fluorescent dyeconjugated to a carbohydrate. Carbohydrates include smaller molecularweight carbohydrates such as D-xylose (MW=150), mannitol (MW=180), andrhamnose (MW=164). D-xylose, a pentose, is the most established of thesugar challenge substances. Humans do not have D-xylose isomerase so theintact molecule is cleared without digestion or degradation, and themechanism of clearance is glomerular filtration. In certain embodiments,the carbohydrate is mannitol.

For compositions comprising a second fluorescent challenge molecule, thesecond molecule may possess different photophysical properties from thefirst challenge molecule. Thus, the two molecules display differentabsorption and emission wavelengths, and allow real-time measurement ofuptake and permeability in the gut. In certain embodiments, the secondfluorescent challenge molecule fluoresces at a wavelength different fromthe first fluorescent challenge molecule.

Methods

The present disclosure provides new methods for assessing gut functionin a subject in need thereof, including the steps of: administering aneffective amount of a fluorescent challenge molecule described hereininto the subject's gut; irradiating the composition absorbed by thesubject's gut with non-ionizing radiation, wherein the radiation causesthe composition to fluoresce; detecting the fluorescence of thecomposition; and assessing gut function in the subject based on thedetected fluorescence and its kinetics of appearance in the blood.

In certain embodiments, the composition is administered orally.

In certain embodiments, the non-ionizing radiation has a wavelength ofat least 350 nm.

In certain embodiments, the composition absorbed by the subject's gut isirradiated and detected in the subject's skin, blood, urine, or fascia.In certain embodiments, the composition absorbed by the subject's gut isirradiated and detected transcutaneously. In certain embodiments, thedetected fluorescence of the fluorescent challenge molecules is measuredover time.

Provided is a method for assessing gut function in a subject in needthereof, comprising the steps of: administering an effective amount of acomposition comprising two fluorescent challenge molecules—a fluorescentchallenge molecule that is not substantially absorbed by a healthy gut,and a second fluorescent challenge molecule is substantially absorbed bya healthy gut—into the subject's gut; irradiating the compositionabsorbed by the subject's gut with non-ionizing radiation, wherein theradiation causes the composition to fluoresce; detecting thefluorescence of each fluorescent challenge molecule in the composition;and assessing gut function in the subject based on the detectedfluorescence. Gut function is assessed by comparing the detectedfluorescence of the fluorescent challenge molecule that is notsubstantially absorbed by a healthy gut to the fluorescent challengemolecule that is substantially absorbed by a healthy gut. The twofluorescent challenge molecules may possess different photophysicalproperties allowing for the simultaneous measurement of the fluorescencethe two molecules.

In certain embodiments, the composition is administered orally. Incertain embodiments, the non-ionizing radiation has a wavelength of atleast 350 nm.

In certain embodiments, the composition absorbed by the subject's gut isirradiated and detected in the subject's skin, blood, urine, or fascia.In certain embodiments, the composition absorbed by the subject's gut isirradiated and detected transcutaneously. In certain embodiments, thedetected fluorescence of the fluorescent challenge molecules is measuredover time.

Also provided is a method of assessing the location of a disease or aninjury in a subject's gut, comprising the steps of: administering aneffective amount of a composition containing a fluorescent challengemolecule that is not substantially absorbed by a healthy gut, and asecond fluorescent challenge molecule is substantially absorbed by ahealthy gut into the subject's gut; irradiating the composition absorbedby the subject's gut with non-ionizing radiation, wherein the radiationcauses the composition to fluoresce; detecting the fluorescence of eachfluorescent challenge molecule in the composition over time; andassessing the location of disease or injury in the subject's gut, basedon the time period between the detected fluorescence of each fluorescentchallenge molecule and administration. Again, the two fluorescentchallenge molecules may possess different photophysical propertiesallowing for the simultaneous measurement of the fluorescence the twomolecules.

In certain embodiments, the composition is administered orally. Incertain embodiments, the non-ionizing radiation has a wavelength of atleast 350 nm.

In certain embodiments, the composition absorbed by the subject's gut isirradiated and detected in the subject's skin, blood, urine, or fascia.In certain embodiments, the composition absorbed by the subject's gut isirradiated and detected transcutaneously.

In certain embodiments, the detected fluorescence of the challengemolecule that is not substantially absorbed by a healthy gut occursbefore or substantially simultaneously as compared the detectedfluorescence of the challenge molecule that is substantially absorbed bya healthy gut—indicating that the disease or injury is located in theproximal portion of the gut. In particular embodiments, this mayindicate the subject is suffering from Celiac disease.

In certain embodiments, the detected fluorescence of the challengemolecule that is not substantially absorbed by a healthy gut occurssubstantially later in time as compared the detected fluorescence of thechallenge molecule that is substantially absorbed by a healthygut—indicating that the disease or injury is located in the distalportion of the gut. In particular embodiments, this may indicate thesubject is suffering from ulcerative colitis.

Also provided is a method of assessing the size of the openings within asubject's gut, comprising the steps of: administering an effectiveamount of a composition comprising at least one fluorescent challengemolecule, wherein each fluorescent challenge molecule has a differentsize, and is absorbed by a healthy gut to a different degree into thesubject's gut; irradiating the composition absorbed by the subject's gutwith non-ionizing radiation, wherein the radiation causes thecomposition to fluoresce; detecting the fluorescence of each fluorescentchallenge molecule in the composition; and assessing the size of theopenings within the subject's gut, based on the size of each fluorescentchallenge molecule.

In certain embodiments, the size of the challenge molecules absorbed bythe subject's gut indicates the minimum size of the openings within thesubject's gut.

Kits

The present disclosure provides a kit for assessing gut function in asubject in need thereof, comprising: a composition comprising afluorescent challenge molecule as described herein, wherein thefluorescent challenge molecule is not substantially absorbed by ahealthy gut, and written instructions for assessing gut function in thesubject, comprising the steps of: administering an effective amount of acomposition of a. into the subject's gut; irradiating the compositionabsorbed by the subject's gut with non-ionizing radiation, wherein theradiation causes the composition to fluoresce; detecting thefluorescence of the composition; and assessing gut function in thesubject based on the detected fluorescence.

In certain embodiments, the instructions include a step foradministering the composition orally.

In certain embodiments, the instructions include a step for irradiatingthe composition with the non-ionizing radiation having a wavelength ofat least 350 nm.

In certain embodiments, the instructions include a step for irradiatingand detecting the composition absorbed by the subject's gut within thesubject's skin, blood, urine, or fascia.

In certain embodiments, the instructions include a step for irradiatingand detecting the composition transcutaneously.

In certain embodiments, the instructions include a step for detectingand measuring fluorescence of the fluorescent challenge molecules overtime.

Provided is a kit for assessing gut function in a subject in needthereof, comprising: a composition comprising two fluorescent challengemolecules—a fluorescent challenge molecule that is not substantiallyabsorbed by a healthy gut, and a second fluorescent challenge moleculeis substantially absorbed by a healthy gut into the subject's gut, andwritten instructions for assessing gut function in the subject,comprising the steps of: administering an effective amount of thecomposition comprising two fluorescent challenge molecules into thesubject's gut; irradiating the composition absorbed by the subject's gutwith non-ionizing radiation, wherein the radiation causes thecomposition to fluoresce; detecting the fluorescence of each fluorescentchallenge molecule in the composition; and assessing gut function in thesubject based on the detected fluorescence.

In certain embodiments, the instructions include a step foradministering the composition orally.

In certain embodiments, the instructions include a step for irradiatingthe composition with the non-ionizing radiation having a wavelength ofat least 350 nm.

In certain embodiments, the instructions include a step for irradiatingand detecting the composition absorbed by the subject's gut within thesubject's skin, blood, urine, or fascia.

In certain embodiments, the instructions include a step for irradiatingand detecting the composition transcutaneously.

In certain embodiments, the instructions include a step for detectingand measuring fluorescence of the fluorescent challenge molecules overtime.

In certain embodiments, the instructions include a step for comparingthe detected fluorescence of the fluorescent challenge molecule that isnot substantially absorbed by a healthy gut to the fluorescent challengemolecule that is substantially absorbed by a healthy gut.

Also provided is a kit for assessing gut function in a subject in needthereof, comprising: a composition comprising two fluorescent challengemolecules—a fluorescent challenge molecule that is not substantiallyabsorbed by a healthy gut, and a second fluorescent challenge moleculeis substantially absorbed by a healthy gut into the subject's gut, andwritten instructions for assessing gut function in the subject,comprising the steps of: administering an effective amount of thecomposition into the subject's gut; irradiating the composition absorbedby the subject's gut with non-ionizing radiation, wherein the radiationcauses the composition to fluoresce; detecting the fluorescence of eachfluorescent challenge molecule in the composition over time; andassessing the location of disease or injury in the subject's gut, basedon the time period between the detected fluorescence of each fluorescentchallenge molecule and administration.

In certain embodiments, the instructions include a step foradministering the composition orally.

In certain embodiments, the instructions include a step for irradiatingthe composition with the non-ionizing radiation having a wavelength ofat least 350 nm.

In certain embodiments, the instructions include a step for irradiatingand detecting the composition absorbed by the subject's gut within thesubject's skin, blood, urine, or fascia.

In certain embodiments, the instructions include a step for irradiatingand detecting the composition transcutaneously.

In certain embodiments, the instructions include a step for detectingand measuring fluorescence of the fluorescent challenge molecules overtime.

Formulation

The compositions of the present disclosure may be administered orally,including by swallowing, so that the compound enters thegastrointestinal tract.

Suitable compositions for oral administration include solid formulationssuch as tablets, lozenges and capsules, which can contain liquids, gels,or powders. Liquid formulations can include solutions, syrups andsuspensions, which can be used in soft or hard capsules. Suchformulations may include a pharmaceutically acceptable carrier, forexample, water, ethanol, polyethylene glycol, cellulose,phosphate-buffered saline (PBS), or an oil. The formulation may alsoinclude one or more emulsifying agents and/or suspending agents.Preparation of pharmaceutically acceptable formulations can beaccomplished according to methods known in the art.

Dosage

Compositions of the present disclosure may be administered in a singledose or in multiple doses to achieve an effective diagnostic objective.After administration, the composition is allowed time to move into thegut, and the selected target site is exposed to light with a sufficientpower and intensity to detect light emanating from the compound withinthe patient's body to provide information that may be utilized by ahealthcare provider (e.g., in making a diagnosis). Doses may vary widelydepending upon, for example, the particular integrated photoactive agentemployed, the areas (e.g., organs or tissues) to be examined, theequipment employed in the clinical procedure, the efficacy of thetreatment achieved, and/or the like. For example, the dosage of thecompound may vary from about 0.1 mg/kg body weight to about 500 mg/kgbody weight in some embodiments. In other embodiments, the dosage of thecompound may vary from about 0.5 to about 2 mg/kg body weight.

Detection and Measurement

After administration, the composition is allowed time to move into thegut, and the selected target site is exposed to light with a sufficientpower and intensity to detect light emanating from the compound withinthe patient's body to provide information that may be utilized by ahealthcare provider (e.g., in making a diagnosis). Detection of thecomposition may be achieved by optical fluorescence, absorbance, and/orlight scattering methods using invasive and/or non-invasive probes suchas endoscopes, catheters, ear clips, hand bands, headbands, surfacecoils, finger probes, and/or the like. Imaging can be achieved usingplanar imaging, optical tomography, optical coherence tomography,endoscopy, photoacoustic technology, sonofluorescence technology, lightscattering technology, laser assisted guided surgery (LAGS), confocalmicroscopy, dynamic organ function monitoring, and/or light scatteringdevices.

Detection and measurement of the fluorescence signal may be determinedas a function of time after delivery of the compositions disclosedherein. Comparison of peak fluorescence signal times may provideinformation regarding the location of any injuries or disease within thegut.

In order that the disclosure described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this disclosure in any manner.

EXAMPLES

Non-limiting examples of challenge molecules include the followingcompounds and pharmaceutically acceptable salts thereof:

Scheme 1 shows non-limiting examples of chemical strategies foractivating pyrazine dyes for conjugation with carbohydrates and othermolecules.

Example 1:3,6-diamino-2,5-bis{N-[(1R)-1-carboxy-2-hydroxyethyl]carbamoyl}pyrazine

This fluorescent challenge molecule exhibits light absorption andemission maxima at 445 nm and 560 nm, respectively. FIG. 3 shows the IVand oral clearance of Example 1, which indicates that there is verylittle oral bioavailability for this relatively high molecular weight(372 g/mol) compound in rats having “normal” gut function. Example 1 maybe categorized as fluorescent challenge molecule is not substantiallyabsorbed by a healthy gut.

Example 2: 3,6-N,N′-bis(2,3-dihydroxypropyl)-2,5-pyrazinedicarboxamide

This fluorescent challenge molecule exhibits light absorption andemission maxima at 486 nm and 600 nm, respectively. FIG. 5 shows themolecule's emission spectra. Example 2 may be categorized as fluorescentchallenge molecule is not substantially absorbed by a healthy gut as themolecular weight is relatively high (344 g/mol).

Example 3: 3,6-diaminopyrazine-2,5-dicarboxylic Acid

The graph shown in FIG. 4 shows the low molecular weight compound clearsfrom the body by the renal system. When the kidneys are ligated, thecompound has no excretion pathway and the fluorescence signal stayselevated because it does not clear. This fluorescent challenge moleculeexhibits light absorption and emission maxima at 396 nm and 536 nm,respectively. Example 3 may be categorized as fluorescent challengemolecule that is substantially absorbed by a healthy gut as themolecular weight is relatively low (198 g/mol).

Example 4: Assessing Gut Function

An exemplary procedure for assessing gut function is as follows: acomposition containing a fluorescent challenge molecule that is notsubstantially absorbed by a healthy gut is administered to a subject.The challenge molecule passes into the subject's gut, and if the gut isinjured or diseased in such a way that increases the gut's permeability,the challenge molecule will be absorbed from the gut into the subject'sbloodstream. While in the blood stream, the fluorescent challengemolecule may be irradiated with non-ionizing radiation, wherein theradiation causes the composition to fluoresce. The fluorescence of anyabsorbed challenge molecule may be detected in the bloodstream; and thegut function may be assessed based on the detected fluorescence. Theappearance of the fluorescent challenge molecule in the bloodstreamindicates the gut has increased permeability.

Another exemplary procedure begins with the administration of acomposition comprising two fluorescent challenge molecules, wherein thefirst fluorescent challenge molecule is not substantially absorbed by ahealthy gut, and the second fluorescent challenge molecule issubstantially absorbed by a healthy gut. The second challenge moleculethat is substantially absorbed by a healthy gut will pass into thesubject's gut, and if the gut is injured or diseased in such a way thatincreases the gut's permeability, the first challenge molecule will beabsorbed from the gut into the subject's bloodstream. While in the bloodstream, the fluorescent challenge molecules may be irradiated withnon-ionizing radiation, wherein the radiation causes the fluorescentchallenge molecules present to fluoresce. The fluorescence of eachabsorbed challenge molecule may be detected in the bloodstream; and thegut function may be assessed by comparing the detected fluorescence ofthe fluorescent challenge molecule that is not substantially absorbed bya healthy gut to the fluorescent challenge molecule that issubstantially absorbed by a healthy gut. The second challenge moleculethat is substantially absorbed by a healthy gut functions as a control,and minimizes effects of digestion kinetics on the results.

The ratio of the first fluorescent challenge molecule that is notsubstantially absorbed by a healthy gut, and the second fluorescentchallenge molecule that is substantially absorbed by a healthy gut isused to assess gut function. A high ratio indicates the gut is highlypermeable, and a low ratio indicates low permeability.

Yet another exemplary procedure may be performed as described above, butin addition, the fluorescence of each absorbed challenge molecule may bedetected in the bloodstream over time; and the location of disease orinjury in the subject's gut may be determined based on the time betweenthe detected fluorescence of each fluorescent challenge molecule andadministration. If the detected fluorescence of the challenge moleculethat is not substantially absorbed by a healthy gut occurs before ornearly the same time as compared the detected fluorescence of thechallenge molecule that is substantially absorbed by a healthy gutindicates that the disease or injury is located in the proximal portionof the gut. If the detected fluorescence of the challenge molecule thatis not substantially absorbed by a healthy gut occurs at nearly the sametime as compared the detected fluorescence of the challenge moleculethat is substantially absorbed by a healthy gut indicates that thedisease or injury is located in the proximal portion of the gut.

The time difference occurs as the composition moves through the gut. Ifthe gut is permeable at the proximal portion, or throughout the gut,both challenge molecules should appear at the same time. If only thedistal portion is permeable, the challenge molecule that is notsubstantially absorbed by a healthy gut should be detected later thanthe challenge molecule that is substantially absorbed by a healthy gut.

The graph shown in FIG. 4 shows the low molecular weight compound clearsfrom the body by the renal system. When the kidneys are ligated, thecompound has no excretion pathway and the fluorescence signal stayselevated because it does not clear. This fluorescent challenge moleculeexhibits light absorption and emission maxima at 396 nm and 536 nm,respectively. Example 3 may be categorized as fluorescent challengemolecule that is substantially absorbed by a healthy gut as themolecular weight is relatively low (198 g/mol).

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present disclosure. However, thedisclosure described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of several aspects of the disclosure. Anyequivalent embodiments are intended to be within the scope of thisdisclosure. Indeed, various modifications of the disclosure in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description, which do not departfrom the spirit or scope of the present inventive discovery. Suchmodifications are also intended to fall within the scope of the appendedclaims.

1-30. (canceled)
 31. A method for assessing gut function in a subject inneed thereof, comprising the steps of: a. administering an effectiveamount of a composition for assessing gut function, the compositioncomprising a fluorescent challenge molecule, wherein the fluorescentchallenge molecule is not substantially absorbed by a healthy gut; b.irradiating the fluorescent challenge molecule absorbed by the subject'sgut with non-ionizing radiation, wherein the radiation causes thefluorescent challenge molecule to fluoresce; c. detecting thefluorescence of the fluorescent challenge molecule; and d. assessing gutfunction in the subject based on the detected fluorescence, wherein thesubject is a human, wherein the fluorescent challenge molecule is apyrazine fluorescent dye, and wherein the fluorescent challenge moleculeabsorbed by the subject's gut is irradiated and detected in thesubject's skin, blood, urine, or fascia.
 32. The method of claim 31,wherein the composition absorbed by the subject's gut is irradiated anddetected transcutaneously.
 33. The method of claim 31, wherein thecomposition absorbed by the subject's gut is irradiated and detected inthe urine of the subject.
 34. The method of claim 31, wherein thedetected fluorescence of the fluorescent challenge molecule is measuredover time.
 35. The method of claim 31, wherein the pyrazine fluorescentchallenge molecule comprises two terminal carboxyl substituents.
 36. Themethod of claim 35, wherein the pyrazine fluorescent challenge moleculefurther comprises two amino substituents.
 37. The method of claim 35,wherein the pyrazine fluorescent challenge molecule further comprisestwo amide substituents.
 38. The method of claim 31, wherein the pyrazinefluorescent challenge molecule comprises two1-carboxy-2-hydroxyethyl]carbamoyl substituents.
 39. The method of claim38, wherein the pyrazine fluorescent challenge molecule furthercomprises two amino substituents.
 40. The method of claim 38, whereinthe pyrazine fluorescent challenge molecule further comprises two amidesubstituents.
 41. The method of claim 31, wherein the pyrazinefluorescent challenge molecule comprises two terminal hydroxypropylsubstituents.
 42. The method of claim 31, wherein the pyrazinefluorescent challenge molecule comprises two amino substituents.
 43. Themethod of claim 31, wherein the pyrazine fluorescent challenge moleculecomprises two amide substituents.
 44. The method of claim 31, whereinthe composition for assessing gut function further comprises at leastone pharmaceutically acceptable carrier, pharmaceutically acceptableemulsifying agent, pharmaceutically acceptable suspending agent or anycombination thereof.
 45. The method of claim 31, wherein the method isnoninvasive for the subject.
 46. The method of claim 31, wherein thecomposition is administered orally.
 47. The method of claim 31, whereinthe non-ionizing radiation has a wavelength of at least 350 nm.